The ability to acquire both accurate and precise qualitative and quantitative mass spectral data in the analysis of complex systems such as proteins is predicated on the ability to measure the physico-chemical attributes of all ions independent of any surrounding matrix.
Successful qualitative analysis requires ensuring that fragment and product ions are correctly aligned or matched to the parent or precursor ions from which they were derived. In order to achieve this level of selectivity, given the large number of ion detections at any given moment of a liquid chromatography separation, an orthogonal pre-ion detection separation technique such as ion mobility separation may be used.
In quantitative experiments accurate and precise measures of abundance by peak volume is a direct consequence of how well the physico-chemical attributes of each eluting compound is measured. Similarly, in qualitative experiments the selectivity of identification relative to the sheer number of possibilities is also a direct consequence of how well these attributes are measured.
It is known to operate a mass spectrometer in either a Data Dependent Acquisition mode of operation or a Data Independent Acquisition mode of operation.
In a Data Dependent Acquisition mode of operation fragment or product ions are formed from parent or precursor ions that are mass resolved (using a quadrupole mass filter isolation window). In contrast, in a Data Independent Acquisition mode of operation fragment or product ions are formed from parent or precursor ions that are either time resolved (MSE) or time and ion mobility drift time resolved (HD-MSE).
With respect to Data Dependent Acquisitions (“DDA”) sensitivity, as defined by accessing the lower end of the experimental dynamic range, is a function of time given there is a minimum parent or precursor ion intensity to produce a sufficient number of fragment or product ions in order to facilitate a valid identification. However, due to the compromising nature of conventional serial Data Dependent Acquisition methods the duty cycle is proportional to the acquisition time and the sensitivity is inversely proportion to duty cycle.
With respect to chimeracy, Data Dependent Acquisition methods utilise an ion selection window whereby only parent or precursor ions present within the selection window are transferred to the collision cell. Sensitivity is inversely proportional to the width of the isolation window. Given the complexity of complex systems in order to retain any semblance of sensitivity rarely, if ever, will there exist just a single parent or precursor ion within an isolation window.
By way of contrast, with Data Independent Acquisition (“DIA”) sensitivity is not compromised given there is no partial peak sampling. Data is acquired on each and every parent or precursor ion throughout its chromatographic elution. However, Data Independent Acquisition methods are prone to suffer from the problem of chimeracy.
The adverse effects of chimeracy can be mitigated through the use of an additional in-line orthogonal ion mobility separation. Accordingly, as with Data Dependent Acquisition methods when analysing complex systems there will normally be a plurality of parent or precursor ions that potentially represent any aligned fragment or product ions. In contrast to Data Dependent Acquisition, the inflexion point is not a function of sensitivity. It is, however, a function of chimeracy.
Both conventional acquisition methods therefore suffer from various problems that limit clarity and depth of coverage in the analysis of complex systems.
In order to further illustrate the problem, the sheer complexity and dynamic range of complex systems will be considered in more detail. The lack of diversity of biomolecules in terms of their elemental composition will also be discussed in more detail below.
A detailed examination of proteins and proteomes reveals a very high degree of similarity in amino acid composition. It is reasonable to assume that on average the number of peptides generated from an enzymatic digestion of a protein is a direct consequence of length. If the amino acid composition is similar and the number of peptides generated is a function of length then it is reasonable to assume that many peptides from multiple proteins will have similar elemental compositions. Similar elemental compositions will restrict the number of available mass to charge ratio values as well as elution space and, if ion mobility spectrometry (“IMS”) is employed then also cross-sectional area.
Overwhelmingly, all bio-molecules are comprised of six elements (C, N, H, O, S, and P). With this being the case, ˜40% of all available space in terms of mass to charge ratio can never be occupied by a biomolecule. More specifically, within a one mass unit window there are four Gaussian distributions each ˜120 mDa wide. Summing the centered count of these distributions reveals that ˜40% of all naturally occurring biomolecules acquired at 40,000 mass resolving power occupy only 10% of the available mass to charge ratio space. In addition, the experimental dynamic range of a typical sample typically spans ˜3-3.5 orders of magnitude. Unfortunately, ˜66.6% of all ion detections reside in the last order of magnitude with ˜50% in the last half order. As such, ˜60% of all ion detections will have similar mass to charge ratios and will also reside at the lowest levels of sensitivity. Given the lack of elemental diversity and the similarity in mass to charge ratio it may be assumed that the distribution of ion detections across a gradient elution follows a Gaussian distribution with the highest density representing the most common elemental composition.
J. Egertson, A. Kuehn, G. Merrihew, N. Bateman, B. MacLean, Y. Ting, J. Canterbury, D. Marsh, M. Kellmann, V. Zabrouskov, C. Wu and M. MacCoss “Multiplexed MS/MS for Improved Data Independent Acquisition” Nat. Methods August (2013) discloses a multiplexing strategy (MSX) wherein five separate 4 m/z isolation windows are analysed per spectrum. These spectra are demultiplexed into the five separate 4 m/z isolation windows using a strategy with similarities to Hadamard multiplexing resulting in data with the sampling frequency of a Data Independent Acquisition approach using 20×20 m/z wide windows but the selectivity of an approach using 100×4 m/z wide windows.
It is desired to provide an improved method of mass spectrometry.